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Microbial Genome Sequencing

Sequencing a microbial genome has never been as easy and affordable as now. We provide a complete sequencing package with the basic bioinformatics analysis included. Our professional services allow you to be hustle-free and focus on your critical tasks.

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Table of Contents

Key Specs
  • NGS only, 799 kr./strain  (€ 109), 499 kr. (€69, ≥ 20 samples)

  • NGS + Nanopore, 2,999 kr./strain (€ 419), 1,999 kr. (€ 289, ≥ 10 samples)

  • Turnaround time: 25-30 workdays on average, max. 40 workdays. Holidays include Denmark, UK, and China.

Workflow
Sample Requirements

For DNA samples submitted, Customer is required to ensure the Quality Control (QC) data at the time of sample submission meets the minimum requirements concerning the total amount, concentration, and quality of the DNA submitted. For microbial culture or direct environmental samples submitted, Customer is obligated to ensure that the sample submitted is non-pathogenic to humans and does not contain any substance that may pose a health risk to the personnel working with the samples in our laboratory.

DNA sample
  • Total quantity ≥ 1.0 μg (short-insert library) or ≥ 5 μg (PCR-free library);

  • Concentration ≥ 20 ng/µL;

  • Purity: A260/280=1.8-2.0.

 

Notes:

(1)  The final concentration of each sample will be measured using QUBIT or an equivalent fluorometer after the samples are received. If the samples do not pass QC, We will notify the Customer, and Customer shall either notify us to cancel the failed samples or re-send the samples concerned within ten workdays. Customer can request that the “QC-failed samples” be sequenced, in which case we will prepare the library and sequence the “QC-failed samples” without bearing any responsibility for the data quality. Customer should notify whether plasmid, chondriosome, chloroplast, and other non-bacterial-origin DNA exist in the samples submitted. Customer should notice that, If the DNA originates from a mixture of multiple microbial species, the quality of final assembly and genome annotation will be compromised significantly, and we will not attempt to separate all assemblies into individual species by any means.

(2)  An aliquot of DNA sample may be stored temporarily in case of a failed library preparation. All DNA samples will be discarded within 3 months after the project is completed.

Microbial pure culture sample

Agar plates: 

colonies should be easily visible and cover > 20% of the surface area on a standard-size petri dish (⌀ 90mm); the agar should not dry out and maintain a good texture for easy collection of cells with a standard sterile loop used in microbiology laboratories.

Pellets: 

Bacterial pellets should be placed in a 1.5 mL or 2 mL centrifuge tube. The size of the pellet shall not be less than that generated by centrifuging 2 mL of E. coli cultures grown in LB overnight at 37 °C, typically covering a round area of ⌀3-5 mm.

Notes:

Customer should pay close attention to contamination on agar plates or in bacterial pellets. Customer must ensure each sample submitted is a single pure culture without being mixed with other species. In any case, we will continue the workflow and sequence the genomic DNA extracted from the samples submitted. We will not accept the complaint on the culture’s purity issue filed by Customer based on the genome sequence data generated in this project.

Environmental sample

The direct submission of soil, lake water, filters, swabs, plant or animal materials, any items or objects colonized by microbes, and other types of environmental samples that contain microbial inhabitants for genomic investigation at our laboratory is subject to individual agreement by email or in other written form between Customer and us concerning the sample type, quantity, and quality before the commence of this agreement.

Deliverables

The project will only commence when we receive the last batch of qualified samples. When a project is successfully completed, we will provide the following items as the standard deliverables:

  • DNA QC report;

  • At least 2 Gb sequence data per sample with base quality of Q20 >95% in fastq format;

  • Contamination and low-quality reads from raw reads are removed before delivery;

  • Basic bioinformatics analysis results, including reads QC, genome assembly, and genome annotation results in excel and fasta formats. The computational work is performed by a machine-supervised automation pipeline w/o manual corrections depending on the Customer’s orders;

  • Project final report.

 

Notes

1) National holidays include those in Denmark, UK, and China; 2) the following sample deliverables may contain items exclusively included in the Pro or Premium service package. 

Sample deliverables
  • DNA QC report
Pricing & Turnaround Time

The rate for a microbial genome with a typical size of 4~6 Mb is 799 kr. (€ 109) per strain and 499 kr. (€69) for ≥ 20 samples sent in a batch. The genome will be sequenced on a short reads platform from either Illumina or MGI. For a larger genome like yeast (~12 Mb) or fungi (10 Mb ~ 200Mb), the rate is calculated as follows: 

            [Standard Rate + 100 kr./Gb x The desired number of Gb]

For example, I recommend an additional 3 Gb of data for a yeast genome, and the rate would be 799 + 100 x 3 = 1,099 kr. Please contact us for a detailed quote on a non-standard project.

A typical bacterial genome can be closed by combining NGS and Nanopore long-read sequencing. The rate for closing a bacterial genome is 2,999 kr./strain (€ 419) and 1,999 kr. (€ 289) for ≥ 10 samples sent in a batch. Please be aware that some bacterial genomes with a large number of long tandem repeats in the genome are difficult to close, even with the best sequencing strategy and practices. 

The project turnaround time is counted from when DNA is ready for sequencing library prep. It is estimated to be 25-30 workdays on average, at most 40 workdays. If the project is not finished within 40 workdays, we will charge no fees related to this project to Customer and refund the prepayment, if any. Please be aware that we are an international team; thus, holidays include Denmark, UK, and China. It is for the contract reason that we set this relatively safe time frame. In most cases, customers will receive the results and final reports within a month, which is our service target.

Add-on Services

The Pro Bioinformatics Package (1,699 kr.; € 229) include all add-on services listed in the workflow. The Premium Bioinformatics Package (9,199 kr.; € 1,299) is highly customer-tailored with the highest level of services provided to address any significant challenges facing customers in their applications; details upon inquiry.

Payment Term

All payments set forth on the invoice are due within 30 days from the date of the invoice. We may impose a service charge on past due amounts between one and one-half percent (1.5%) and three percent (3%) per month, not to exceed the maximum amount permitted by law. Upon the Customer’s request, we will provide a statement within ten days.

The service agreement should be governed by and construed in accordance with the law in Denmark.

Service Contract

Draft Agreement

References

Tatusova, T., DiCuccio, M., Badretdin, A., Chetvernin, V., Nawrocki, EP, Zaslavsky, L., … & Ostell, J. (2016). NCBI prokaryotic genome annotation pipelineNucleic acids research , 44 (14), 6614-6624.

Wick, R. R., Judd, L. M., Gorrie, C. L., & Holt, K. E. (2017). Unicycler: resolving bacterial genome assemblies from short and long sequencing readsPLoS computational biology13(6), e1005595.

Zeng, Y., Chen, X., Madsen, A. M., Zervas, A., Nielsen, T. K., Andrei, A. S., … & Hansen, L. H. (2020). Potential rhodopsin-and bacteriochlorophyll-based dual phototrophy in a high Arctic glaciermBio11(6), e02641-20.

Zeng, Y., Wu, N., Madsen, A. M., Chen, X., Gardiner, A. T., & Koblížek, M. (2021). Gemmatimonas groenlandica sp. nov. is an aerobic anoxygenic phototroph in the phylum Gemmatimonadetes. Frontiers in Microbiology11, 606612.

Cai, H., McLimans, C. J., Beyer, J. E., Krumholz, L. R., & Hambright, K. D. (2023). Microcystis pangenome reveals cryptic diversity within and across morphospeciesScience Advances9(2), eadd3783.

Bioinformatics
Tools Frequently Used:

Unicycler, CD-HIT, Quast, Trimmomatic, Bowtie2, Diamond, Salmon, FastQC, NCBI PGAP, BLASTn, MAFFT,  FastANI, CompareM, EasyFig, Circa, RAST server, PhyloPhlAn, FastTree, iTOL, GTDB-Tk, Prokka, RefineM, CheckM, etc.

Here is the collection of scripts and Linux commands.

FAQ
Q: Can I close a bacterial genome only with short reads by applying an extremely high sequencing depth, say 1,000x?

You can’t. No matter how much you sequence the genome with short reads methods, you won’t generate a closed genome. Short reads cannot span the repetitive regions longer than the length of the reads themselves. Repetitive sequences are widespread in microbial genomes due to genome evolution. Thus, there are always gaps in short reads-assembled genomes. Most of the gaps are located in the regions containing transposons, phage-related sequences, or genes of recombinases or integrases.

Q: Can Nanopore sequencing technology replace short reads sequencing platforms?

Maybe in the not-far future but not present. Although Nanopore reads’ accuracy is improving, it is still way below that of short reads generated from Illumina or MGI sequencers. New developments put Nanopore reads into the Q30 category but only on a limited fraction of reads at a small scale and relying on expensive chemistries. The cost-effectiveness of short reads is unmatched. Short NGS reads are often used to polish erroneous Nanopore long reads. Combining Nanopore’s long reads and NGS’s short reads is the best strategy to close a small genome with very high accuracy.

Q: Does your workflow work for yeast, fungi or micro-eukaryotes?

It is not guaranteed. This workflow was designed using a small bacterial or archaeal genome as a reference. For a larger genome, the sequencing depth should be accordingly higher. Usually, we recommend 50-100x long reads and 200-500x short reads to generate a good hybrid assembly. The more complex a genome is, the more reads are desired. Our experience shows it’s much harder to close a yeast/fungal/micro-eukaryote’s genome than a bacterial genome.

Q: Do you use any proprietary software for bioinformatic analysis?

No. We only use open-source programs to ensure the reproducibility of our results. All protocols are publicly available on our website, or customers will be referred to original scientific papers. We aim to make all protocols available on our websites and keep them updated over time by following the latest literature.


See more at mBioWorks FAQ.

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